Staphylococcus aureus is an opportunistic bacterial pathogen responsible for a diverse spectrum of human and animal diseases. Although 11 putative capsular serotypes have been reported, types 5 and 8 constitute about75 percent of clinical isolates. The type 5 (CP5) and 8 (CP8) polysaccharides are structurally very similar to each other; they differ only in the linkages between the sugars and in the sites of O-acetylation. The overall goal of this study is to use a molecular approach to define the functions, relative biological activity, and regulation of S. aureus capsule genes. The molecular events controlling the biosynthesis of CP5 and CP8 will be investigated, and the existence and prevalence of other capsular serotypes will be examined. In the first specific aim, we will determine the enzymatic functions of type 5 capsule genes that are involved in amino sugar biosynthesis: cap5D, cap5E, cap5F, cap5G, and cap5N. We will express the recombinant proteins in Escherichia coli and conduct in vitro assays to evaluate the enzymatic functions of the purified proteins. The second aim will address the biological differences between strains producing type 5 or 8 capsules. We plan to replace the serotype 5-specific capsule genes (cap5HIJK) of strain Reynolds with the serotype 8-specific genes (cap8HIJK) of strain Becker so that strain Reynolds will produce CP8. The isogenic serotype 5/8 strains will then be compared for quantity of capsule produced, resistance to killing in an in vitro opsonophagocytic killing assay, and virulence in a mouse model of bacteremia and renal abscess formation. The influence of capsule production on the deposition of complement on S. aureus cells will also be explored. The third specific aim will examine strains of S. aureus that do not react with antibodies to capsule type 1,2,5, or 8. Since most of these "non-typeable" strains carry the genes for CP5 or CP8 production, we will determine why capsule is not expressed. We also plan to evaluate non-typeable isolates for the production of capsules other than serotypes 1, 2, 5, and 8. The proposed studies will lead to a better understanding of the organization, structure, function and regulation of capsule expression in S. aureus.